RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) is used to silence gene expression. RNAi is induced by short (i.e. <30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell (Fire et al., 1998, Nature 391:806-811). These short dsRNA molecules called “short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs (“mRNAs”) which share sequence homology with the siRNA (Elbashir et al., 2001, Genes Dev, 15:188-200). It is believed that one strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation. The siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
RNAi provides a very exciting approach to treating and/or preventing diseases. Some major benefits of RNAi compared with various traditional therapeutic approaches include: the ability of RNAi to target a very particular gene involved in the disease process with high specificity, thereby reducing or eliminating off target effects; RNAi is a normal cellular process leading to a highly specific RNA degradation; and RNAi does not trigger a host immune response as in many antibody based therapies.
Several interfering RNA delivery methods are being tested/developed for in vivo use. For example, siRNAs can be delivered “naked” in saline solution; complexed with polycations, cationic lipids/lipid transfection reagents, or cationic peptides; as components of defined molecular conjugates (e.g., cholesterol-modified siRNA, TAT-DRBD/siRNA complexes); as components of liposomes; and as components of nanoparticles. These approaches have shown varying degrees of success. Thus, there remains a need for new and improved methods for delivering siRNA molecules in vivo to achieve and enhance the therapeutic potential of RNAi.
Several cell-penetrating peptides (CPPs) or membrane permeant peptides (MPPs) have been described (Jarver and Langel, 2004, Drug Discov Today 9:395-402) as conjugates to deliver peptides into cells. Of these, the protein transduction domain (PTD) of the HIV-1 TAT protein appears to be particularly effective. The TAT peptide has been used to deliver biologically active cargo to cells in vitro and in vivo (Wadia et al., 2003, Curr Protein Pept Sci. 4:97-104; Bullok K E et al., 2006, Mol Imaging 5:1-15). Dowdy's group also demonstrated that co-delivery of the HA2 peptide enhances release of cargo from macropinosomes following TAT-mediated delivery (Wadia et al., 2004, Nat Med. 10:310-315).
Several groups have explored the use of CPPs to deliver siRNA (reviewed in Meade and Dowdy, 2007, Adv Drug Deliv Rev. 59:134-140). This review points out the problems associated with direct attachment of siRNA to TAT. The protein transduction domain (PTD) of the HIV-1 TAT protein is a CPP that appears to be particularly effective. The TAT peptide has been used to deliver biologically active cargo to cells in vitro and in vivo (Wadia and Dowdy, 2003, Curr Protein Pept Sci. 4:97-104; Bullok et al., 2006, Mol. Imaging 5:1-15). The addition of 10 histidine residues (10H) to TAT has been shown to facilitate endosomal release of DNA (Lo & Wang, Biomaterials 29:2408-2414, 2008).
While several groups have explored the use of CPPs to deliver interfering RNA molecules (See Meade and Dowdy, 2007, Adv Drug Deliv Rev. 59:134-140), the main challenge to this approach involves linking the interfering RNA to the CPPs while maintaining the ability of the complex to interact with and enter the intracellular environment. In particular, the negative charge of the interfering RNA neutralizes the positively charged CPPs, which renders such complexes incapable of cellular delivery. Thus, there remains a need to identify effective means for delivering interfering RNA molecules to target cells.